Methods, reagents and kits for detecting minimal residual disease

ABSTRACT

The invention relates to the field of minimal residual disease (MRD) diagnostics, which is progressively more applied for the evaluation of treatment effectiveness in patients with a hematological malignancy, such as B-cell precursor acute lymphoblastic leukemia (BCP-ALL), B-cell chronic lymphocytic leukemia (B-CLL), and multiple myeloma (MM). Provided are unique reagent compositions with carefully selected and thoroughly tested combinations of antibodies, for ≥8-color flow cytometric stainings as well as for 10-color and 12-color flow cyometric stainings, which can reach sensitivities of at least 10−4, even down to 10−5. Also provided are diagnostic kits and methods for detecting MRD.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 15/812,384, filed Nov. 14, 2017 (pending), which is a divisional of U.S. patent application Ser. No. 14/407,268, filed Dec. 11, 2014 (abandoned), which is a 371 National Stage application of International Application No. PCT/NL2013/050420, filed Jun. 14, 2013, published in English, which claims the benefit of U.S. Provisional Application Ser. No. 61/659,524, filed on Jun. 14, 2012, each of which are incorporated herein by reference in their entirety.

The invention relates to the field of cancer diagnosis, more specifically to means and method for the monitoring of disease development during and after treatment or for the detection of minimal disseminated disease. Cytostatic or cytotoxic treatment induces remission in the majority of patients with lymphoid malignancies. Nevertheless many of these patients relapse. Apparently the current cytostatic or cytotoxic treatment protocols are not capable of killing all malignant cells in these relapsing patients, although they reached so-called complete remission according to cytomorphological criteria. Since the detection limit of cytomorphological techniques is not lower than 1-5% malignant cells, it is obvious that such techniques can only provide superficial information about the effectiveness of treatment, up to 10¹⁰ tumor cells still potentially remaining in the body

Techniques with a higher sensitivity to detect “minimal residual disease” or minimal disease (MRD) are needed to obtain better insight in the reduction of tumor mass during induction treatment and further eradication of the malignant cells during maintenance treatment from one or more than one tissue. The application of flow cytometry for detection of MRD is traditionally based on discrimination between malignant cells and normal leukocytes via malignancy-associated phenotypic characteristics, such as aberrant expression, overexpression, and cross-lineage expression of antigens.

Current 4-color and 6-color flow cytometry reaches a fair sensitivity of 10⁻³ (to 10⁻⁴) in most patients with a hematological malignancy. However, it should be noted that the detection of low frequencies of malignant cells in blood, bone marrow and other body fluids such as cerebrospinal fluid during and after therapy and after hematopoietic stem cell transplantation can be hampered by high frequencies of normal regenerating cells. The extent and the pattern of regeneration differs per treatment protocol, per phase of treatment, per time of sampling, and seems to be dependent on the intensity of the preceding treatment: the more intensive the treatment, the more prominent the regeneration of hematopoietic cells.

Logically, both the background of regenerating cells and the drug-induced immunophenotypic shifts reduce the sensitivity and specificity of the existing 4-color and 6-color flow cytometric MRD methods. This has mainly lead to approaches in which either multiple combinations of markers are used to evaluate MRD in a patient or, alternatively to use one or a few patient-specific combinations of markers. Recognizing the need for improved diagnostic methods for MRD, the present inventors set out to identify additional markers which could be used to obtain a more sensitive and reliable assay for detecting MRD, particularly based on a fully integrated approach, in which information of multiple markers is combined via multivariate analysis. In addition, this new approach is not anymore limited to individual patients, but is applicable to every patient of a specific disease category, such B-cell precursor acute lymphoblastic leukemia (BCP-ALL), B-cell chronic lymphocytic leukemia (B-CLL) and multiple myeloma (MM).

After careful selection of the relevant markers, design of appropriate combinations of antibodies in multi-color tubes, and the selection of suited fluorochromes (based on need for brightness, compensation, stability, etc.), a set of antibody reagents was developed. The studies were complemented with extensive multicentric evaluation of the consensus panels in order to reshape and achieve an optimal efficiency. The inventors designed novel≥8-color stainings with carefully selected and thoroughly tested combinations of antibodies, which can reach sensitivities of 10⁻⁴ to 10⁻⁵ Based on design-testing-redesign-retesting-redesign (etc.), specific combinations of fluorochrome-conjugated antibodies have been developed per disease category, such as BCP-ALL, B-CLL and MM. One or two≥8-color combinations per patient will allow careful MRD monitoring with sensitivities of at least 10⁻⁴. The provided 10-color and 12-color antibody combinations can even better discriminate between normal cells and their malignant counterparts, thereby allowing for MRD detection with sensitivities down to 10⁻⁵.

Here we present novel 8-color 10-color and 12-color antibody combinations for detection of MRD in a sample, e.g. blood or bone marrow, isolated from patients with:

-   -   B-cell precursor acute lymphoblastic leukemia (BCP-ALL)     -   B-cell chronic lymphocytic leukemia (B-CLL) or     -   Multiple myeloma (MM) and related plasma cell disorders (PCD).

These multi-color immunostainings can be performed according to the so-called EuroFlow protocols as described by Van Dongen et al. Leukemia 2012; 26: 1908-1075 and by Kalina et al. Leukemia 2012; 26: 1986-2010.

Accordingly, the invention provides unique reagent compositions for flow cytometric detection of MRD, comprising a combination of at least eight distinct fluorochrome-conjugated antibodies. In particular, the reagent compositions are of use for detecting MRD in patients with BCP-ALL, B-CLL or MM/PCD. In a preferred embodiment, the composition comprises monoclonal antibodies against a given CD antigen. CD stands for cluster designation and is a nomenclature for the identification of specific cell surface antigens or intracellular antigens defined by monoclonal antibodies. (Monoclonal) antibodies against the indicated markers can be commercially obtained from various companies, including Becton/Dickinson (BD) Biosciences, Dako, Beckman Coulter, CYTOGNOS, Caltag, Pharmingen, Exbio, Sanquin, Invitrogen, and the like.

Flow Cytometric MRD Detection in BCP-ALL

In one embodiment, the invention provides a reagent composition for flow cytometric detection of BCP-ALL cells in a human subject, comprising a panel of at least eight distinct fluorochrome-conjugated antibodies. The BCP-ALL panel comprises antibodies against the four “core markers” CD10, CD19, CD20, CD34 and CD45.

Preferably, the panel further comprises one or more antibodies selected from the group of antibodies against CD38, CD81, CyIgμ, and deoxynucleotidyl transferase (NuTdT). Very good results are obtained if the panel further comprises one or more sets of antibodies selected from (a) set of antibodies against CD66c and CD123; (b) set of antibodies against CD304 and CD73; and (c) set of antibodies against SmIgκ and SmIgλ, wherein the antibodies within each set are conjugated to the same fluorochrome. In a specific aspect, the BCP-ALL panel comprises antibodies against CD10, CD19, CD20, CD34, CD45, one or more antibodies selected from the group of antibodies against CD38, CD81, CyIgμ, NuTdT, and two or more sets of antibodies selected from (a) set of antibodies against CD66c and CD123; (b) set of antibodies against CD304 and CD73; and (c) set of antibodies against SmIgκ and SmIgλ, wherein the antibodies within each set are conjugated to the same fluorochrome.

For example, a reagent composition comprises distinct fluorochrome-conjugated antibodies directed against one of the following combinations of markers:

-   -   (i) CD20, CD45, CD81, CD66c, CD123, CD34, CD19, CD10 and CD38,         wherein the antibodies against CD66c and CD123 are conjugated to         the same fluorochrome;     -   (ii) CD20, CD45, CD81, CD304, CD73, CD34, CD19, CD10 and CD38,         wherein the antibodies against CD304 and CD73 are conjugated to         the same fluorochrome;     -   (iii) CD20, CD45, NuTdT, SmIgκ, SmIgλ, CyIgμ, CD19, CD34 and         CD10, wherein the antibodies against SmIgκ and SmIgλ are         conjugated to the same fluorochrome. See for instance the         8-color BCP-ALL MRD Panel 1A.

As another example, it comprises distinct fluorochrome-conjugated antibodies directed against the markers CD20, CD45, CD81, NuTdT, CD34, CD19, CD10 and CD38, and one or more sets of antibodies selected from (a) set of antibodies against CD66c and CD123; (b) set of antibodies against CD304 and CD73; and (c) set of antibodies against SmIgκ and SmIgλ, wherein the antibodies within each set are conjugated to the same fluorochrome. See for instance the 10-color tube in Panel 1B comprising antibodies against the markers CD20, CD45, CD81, NuTdT, CD66c, CD123, CD304, CD73, CD34, CD19, CD10 and CD38.

In a further specific aspect, the composition comprises a combination of fluorochrome-conjugated antibodies directed against the markers CD20, CD45, CD81, NuTdT, CD66c, CD123, CD304, CD73, SmIgκ, SmIgλ, CyIgμ, CD34, CD19, CD10 and CD38, wherein the antibodies against each of the sets CD66c/CD123, CD304/CD73 and SmIgκ/SmIgλ are conjugated to the same fluorochrome. See for instance the 12-color tube in panel 1C.

Suitable fluorochromes for conjugating antibodies for use in the present invention against the recited markers are known in the art. As will be understood, the fluorochromes used within a reagent composition should be distinguishable from each other by flow cytometry. The fluorochromes are preferably selected for brightness, limited spectral overlap and limited need for compensation, stability, etc (see: Kalina et al. Leukemia 2012: 26: 1986-2010).

The following panel of fluorochromes is of particular use in a BCP-ALL reagent composition according to the invention: (1) pacific blue (PacB), brilliant violet 421 (BV421) or Horizon V450, (2) pacific orange (PacO), Horizon V500 (HV500), BV510, Khrome orange (KO) or 00515, (3) fluorescein isothiocyanate (FITC) or Alexa488, (4) phycoerythrin (PE), (5) peridinin chlorophyl protein/cyanine 5.5 (PerCP-Cy5.5), PerCP or PE-TexasRed, (6) phycoerythrin/cyanine7 (PE-Cy7), (7) allophycocyanine (APC) or Alexa647, and (8) allophycocyanine/hilite 7 (APC-H7), APC-Cy7, Alexa680, APC-A750, APC-C750 or Alexa700. After multiple testing rounds, the present inventors observed that very good results can be obtained if the following fluorochromes are chosen: Pacific Blue, brilliant violet 421 or Horizon V450, PacO or Horizon V500, FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, and APC-H7 or APC-A750 or APC-C750. In a specific aspect, the invention provides for a reagent composition shown in Table 1, panel 1A, panel 1B or panel 1C.

TABLE 1 Exemplary reagent compositions for MRD detection in BCP-ALL.. Panel 1A. Marker Composition of 8-color BCP-ALL MRD panels of the invention tube PacB PacO FITC PE PerCPCy5.5 PECy7 APC APCC750 1 CD20 CD45 CD81 CD66c and CD34 CD19 CD10 CD38 CD123 2 CD20 CD45 CD81 CD304 and CD34 CD19 CD10 CD38 CD73 2 CD20 CD45 NuTdT SmIgκ and CyIgμ CD19 CD34 CD10 SmIgλ Panel 1B. Marker Composition of 10-color BCP-ALL MRD panel of the invention F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 CD20 CD45 CD81 NuTdt CD66c CD304 CD34 CD19 CD10 CD38 and and CD123 CD73 Panel 1C. Marker Composition of 12-color BCP-ALL MRD panel of the invention F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 CD20 CD45 CD81 NuTdt CD66c CD304 SmIgκ CyIgμ CD34 CD19 CD10 CD38 and and CD73 SmIgλ

Flow Cytometric MRD Detection in B-CLL

In another embodiment, the invention provides a reagent composition for flow cytometric detection of B-cell chronic lymphocytic leukemia (B-CLL) in a human subject, comprising a panel of at least eight distinct fluorochrome-conjugated antibodies, the panel comprising at least antibodies against the seven “core markers” CD5, CD27, CD79b, CD3, CD200, CD81 and CD19. It was found that CD22 and/or Receptor tyrosine kinase-like orphan receptor 1 (ROR1) can be used as valuable additional marker(s). Very good results were obtained in combination with the markers CD43 and CD38.

Preferred marker combinations for detecting B-CLL are as follows:

(a) CD27, CD3, CD79b, CD5, CD22, CD19, CD200 and CD81

(b) CD5, CD3, CD79b, ROR1, CD27, CD19, CD200 and CD81

(c) CD27, CD3, CD79b, ROR1, CD5, CD22, CD19, CD20, CD200 and CD81

(d) CD27, CD3, CD79b, ROR1, CD5, CD22, CD19, CD20, CD200, CD43, CD81 and CD38.

The following panel of fluorochromes is of particular use in a CLL reagent composition according to the invention: (1) pacific blue (PacB), brilliant violet 421 (BV421) or Horizon V450, (2) pacific orange (PacO), Horizon V500 (HV500), BV510, Khrome orange (KO) or 00515, (3) fluorescein isothiocyanate (FITC) or Alexa488, (4) phycoerythrin (PE), (5) peridinin chlorophyl protein/cyanine 5.5 (PerCP-Cy5.5), PerCP or PE-TexasRed, (6) phycoerythrin/cyanine7 (PE-Cy7), (7) allophycocyanine (APC) or Alexa647, and (8) allophycocyanine/hilite 7 (APC-H7), APC-Cy7, Alexa680, APC-A750, APC-C750 or Alexa700. After multiple testing rounds, the present inventors observed that very good results can be obtained if the following fluorochromes are chosen: Pacific Blue, brilliant violet 421 or Horizon V450, PacO or Horizon V500, FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, and APC-H7 or APC-A750 or APC-C750.

In a specific aspect, the invention provides for a reagent composition shown in Table 2.

TABLE 2 Exemplary reagent compositions for MRD detection in BC-CLL Panel 2A. Composition of 8-color CLL MRD panel BV421 BV510 FITC PE PerCPCy5.5 PECy7 APC APCC750 CD27 CD3 CD79b CD5 CD22 CD19 CD200 CD81 CD5 CD3 CD79b ROR1 CD27 CD19 Cd200 CD81 Panel 2B. Composition of 10-color CLL MRD tube F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 CD27 CD3 CD79b ROR1 CD5 CD22 CD19 CD20 CD200 CD81 Panel 2C. Composition of 12-color CLL MRD tube F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 CD27 CD3 CD79b ROR1 CD5 CD22 CD19 CD20 CD200 CD43 CD81 CD38

Flow Cytometric MRD Detection in Multiple Myeloma/Plasma Cell Disease (MM/PCD)

A still further aspect of the invention relates to a reagent composition for detecting MM or PCD cells. The panel comprises antibodies against the four “core markers” CD138, CD38, CD56 and CD19, supplemented with at least four additional markers selected from the group consisting of CD27, CD117, CD81, CD229, CD45, CyIgκ and CyIgλ, CD45 is a preferred fifth marker, preferably in combination with CD27, CD117 and CD81 or CD229, CyIgκ and CyIλ.

Provided is a reagent composition with distinct fluorochrome-conjugated antibodies against either one of the following panels of markers:

(n) CD45 CD CD56 CD27 CD19 CD117CD81

(o) CD45 CD138CD38 CD56 CD229CD19 CyIgκ CyIgλ

(p) CD138 CD27 CD38 CD56 CD45 CD19 CD117CD81

(q) CD138 CD27 CD38 CD56 CD229CD19 CyIgκ CyIgλ

(r) CD138 CD 27 CD38 CD56 CD45 CD19 CyIgκ CyIgλ

For example, provided is a reagent composition for flow cytometric detection of MM or PCD in a human subject, comprising a panel of at least eight distinct fluorochrome-conjugated antibodies, the panel comprising at least antibodies against the core markers CD138, CD38, CD56 and CD19, supplemented with at least four additional markers selected from the group consisting of CD27, CD117, CD81, CD229, CD45, CyIgκ and CyIgλ. Preferably, CD45 is the fifth marker, more preferably in combination with the markers CD27, CD117 and CD81, or in combination with the markers CD229, CyIgκ and CyIgλ. Preferred reagent compositions comprise distinct fluorochrome-conjugated antibodies directed against one of the following combinations of markers:

(iv) CD45, CD138, CD38, CD56, CD27, CD19, CD117 and CD81

(v) CD45, CD138, CD38, CD56, CD229, CD19, CyIgκ and CyIgλ

(vi) CD138, CD27, CD38, CD56, CD45, CD19, CD117 and CD81

(vii) CD138, CD27, CD38, CD56, CD229, CD19, CyIgκ and CyIgλ

(viii) CD138, CD27, CD38, CD56, CD45, CD19, CyIgκ and CyIgλ

See for instance the 8-color PCD MRD Panel 3A.

Very good results were obtained using fluorochrome-conjugated antibodies directed against the markers CD138, CD27, CD38, CD56, CD45, CD19, CD117, CD81 and one or both set(s) of antibodies selected from (a) set of antibodies against CD229 and CD28; and (b) set of antibodies against CyIgκ and CyIλ. See for instance the 10-color tube in Panel 3B and the 12-color tube in Panel 3C.

The following panel of fluorochromes is of particular use in a MM/PCD reagent composition according to the invention: (1) pacific blue (PacB), brilliant violet 421 (BV421) or Horizon V450, (2) pacific orange (PacO), Horizon V500 (HV500), BV510, Khrome orange (KO) or 00515, (3) fluorescein isothiocyanate (FITC) or Alexa488, (4) phycoerythrin (PE), (5) peridinin chlorophyl protein/cyanine 5.5 (PerCP-Cy5.5), PerCP or PE-TexasRed, (6) phycoerythrin/cyanine7 (PE-Cy7), (7) allophycocyanine (APC) or Alexa647, and (8) allophycocyanine/hilite 7 (APC-H7), APC-Cy7, Alexa680, APC-A750, APC-C750 or Alexa700. After multiple testing rounds, the present inventors observed that very good results can be obtained if the following fluorochromes are chosen: Pacific Blue, brilliant violet 421 or Horizon V450, PacO or Horizon V500, FITC, PE, PerCP-Cy5.5, PE-Cy7, APC, and APC-H7 or APC-A750 or APC-C750.

TABLE 3 Exemplary reagent compositions for MRD detection in MM/PCD Panel 3A. Composition of PCD MRD panel PacB APCH7 or or BV421 HV500 APCA750 or or or Tube HV450 PacO FITC PE PerCPCy5.5 PECy7 APC APCC750 1 CD45 CD138 CD38 CD56 CD27 CD19 CD117 CD81 2 CD45 CD138 CD38 CD56 CD229 CD19 CyIgκ CyIgλ 3 CD138 CD27 CD38 CD56 CD45 CD19 CD117 CD81 4 CD138 CD27 CD38 CD56 CD229 CD19 CyIgκ CyIgλ 5 CD138 CD27 CD38 CD56 CD45 CD19 CyIgκ CyIgλ Panel 3B. Composition of 10-color PCD MRD panel F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 CD138 CD27 CD38 CD56 CD45 CD19 CD117 CD81 CD229 CD288 CD138 CD27 CD38 CD56 CD45 CD19 CD117 CD81 CyIgκ CyIgλ Panel 3C. Composition of 12-color PCD MRD panel F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 CD138 CD27 CD38 CD56 CD45 CD19 CD117 CD81 CD229 CD28 CyIgκ CyIgλ

A further aspect of the invention relates to a diagnostic kit for detecting MRD, in particular BCP-ALL MRD, CLL MRD or MM/PCD MRD comprising one or more of the reagent compositions described herein above, optionally together with instructions for use, buffer, and/or control samples (see: Kalina et al. Leukemia 2012: 26: 1986-2010). In one embodiment, there is provided a BCP-ALL kit comprising one or more reagent compositions of Table 1. In another embodiment, there is provided a CLL kit comprising one or more reagent compositions of Table 2. In still a further embodiment, there is provided a PCD kit comprising one or more reagent compositions of Table 3.

The invention also relates to a method for flow cytometric detection of MRD, comprising the steps of providing a biological sample from a human subject and contacting at least a portion (aliquot) of the sample with a reagent composition provided herein. Any type of sample known or suspected to contain leukocytes may be used directly, or after lysing non-nucleated red cells, or after density centrifugation, or after cell sorting procedures. For example, the sample is peripheral blood, bone marrow, tissue sample such as lymph nodes, adenoid, spleen, or liver, or other type of body fluid such as cerebrospinal fluid, vitreous fluid, synovial fluid, pleural effusions or ascitis. Peripheral blood or bone marrow is preferred.

Provided is a multi-color flow cytometric method for detecting minimal residual disease (MRD) in a biological sample comprising cells, preferably B-lineage cells (B-cell precursors, B-lymphocytes, and plasma cells), comprising the steps of:

(i) staining the sample with a reagent composition according to the invention,

(ii) subjecting the sample to flow cytometry; (iii) gating on cells for expression of the selected markers detected by the antibodies present in the reagent composition;

(iv) distinguishing between normal and malignant cells, based on the expression profile of the multiple markers.

Preferably, the analysis in step (iv) involves multivariate analysis, preferably principal component analysis (PCA), wherein each marker has added value in the distinction process via the principal component analysis. Advantageously, using automated population separation—APS view—is used employing for example Infinicyt software, or multidimensional scaling (MDS) analysis.

PCA is a mathematical procedure that uses an orthogonal transformation to convert a set of observations of possibly correlated variables into a set of values of uncorrelated variables called principal components. The number of principal components is less than or equal to the number of original variables. This transformation is defined in such a way that the first principal component has as high a variance as possible (that is, accounts for as much of the variability in the data as possible), and each succeeding component in turn has the highest variance possible under the constraint that it be orthogonal to (uncorrelated with) the preceding components. Principal components are guaranteed to be independent only if the data set is jointly normally distributed. PCA is sensitive to the relative scaling of the original variables. Depending on the field of application, it is also named the discrete Karhunen-Loève transform (KLT), the Hotelling transform or proper orthogonal decomposition (POD). Alternatively to PCA, MDS or any other type of well-established multivariate analysis, can be used (see: Pedreira et al. Trends Biotechnol 2013).

In one embodiment, there is provided is a multi-color flow cytometric method for detecting minimal residual disease (MRD) in a biological sample comprising lymphocytes, wherein MRD is BCP-ALL, comprising the steps of:

(i) staining the sample with a BCP-ALL reagent composition according to the invention, preferably a composition selected from any one of panels 1A, 1B or 1C;

(ii) subjecting the sample to flow cytometry; (iii) gating on mature B cells and BCP-cells for expression of the markers detected by the antibodies present in the reagent composition; (iv) distinction between normal and malignant BCP cells, based on the application of multiple markers, each having added value in the distinction process via the principal component analysis. See examples in Example 1 and FIGS. 1 and 2 for an exemplary analysis allowing for detection of MRD in BCP-ALL patients.

In another embodiment, there is provided is a multi-color flow cytometric method for detecting minimal residual disease (MRD) in a biological sample comprising lymphocytes, wherein MRD is CLL, comprising the steps of:

(i) staining the sample with a CLL reagent composition according to the invention, preferably a composition selected from any one of panels 2A, 2B or 2C;

(ii) subjecting the sample to flow cytometry; (iii) gating B-lymphocytes for expression of the markers detected by the antibodies present in the reagent composition; (iv) distinction between normal and malignant B cells, based on the application of multiple markers, each having added value in the distinction process via the principal component analysis.

See examples in Example 2 and FIGS. 3 and 4 for an exemplary analysis allowing for detection of MRD in CLL patients.

In yet another embodiment, there is provided is a multi-color flow cytometric method for detecting minimal residual disease (MRD) in a biological sample comprising lymphocytes, wherein MRD is MM/PCD, comprising the steps of:

(i) staining the sample with a MM/PCD reagent composition according to the invention, preferably a composition selected from any one of panels 3A, 3B or 3C;

(ii) subjecting the sample to flow cytometry; (iii) gating plasma cells for expression of the markers detected by the antibodies present in the reagent composition; (iv) distinction between normal and malignant plasma cells, based on the application of multiple markers, each having added value in the distinction process via the principal component analysis.

See examples in Example 3 and FIGS. 5 and 6 for an exemplary analysis allowing for detection of MRD in MM/PCD patients.

LEGEND TO THE FIGURES

FIG. 1 . Typical example of how to use the CD19 and CD45 identification markers in combination with SSC (Panel A to C) for the distinction between BCP cells and other nucleated cells in a bone marrow sample from a BCP-ALL patient during therapy. In each plot, light grey events correspond to non-B cells in the sample, while dark grey events are mature B-cells and black events BCP cells. In the multivariate analysis representation (APS1) of principal component 1 vs. principal component 2 (Panel D), both mature B cells and BCP cells are clearly separated from all other events based on all informative parameters (e.g. CD19, CD45, SSC).

FIG. 2 .—Illustrating example of how to use immunophenotypic characterization markers CD10, CD20, CD34, CD66c/CD123, and CD38 in combination with SSC for the distinction between BCP-ALL cells and normal residual B-cells in a bone marrow sample from a BCP-ALL patient during therapy (Panels A to D). Only bone marrow B-cells are displayed, after gated/selected as described in FIG. 1 . In each plot, black dots correspond to BCP-ALL cells in the sample, while grey dots are normal B-cells. Compared to normal B-cells, BCP-ALL cells show overexpression of CD81 (Panel D), CD10 (Panels A and D), and CD66c/CD123 (Panel C). In the APS1 (principal component 1 versus principal component 2) representation based on all immunophenotypic markers and scatter characteristics evaluated (Panel E), normal residual B-cells (grey) are clearly discriminated from BCP-ALL cells (black).

FIG. 3 .—Illustrating example of how to use the CD19 and CD3 identification markers in combination with SSC (Panels A to C) for the distinction between mature B-cells and other nucleated cells in a peripheral blood sample from a CLL patient. In each plot, grey events correspond to non-B-cells in the sample, while black events are total peripheral blood B-cells. In the multivariate analysis representation (APS1) of principal component 1 vs. principal component 2 (Panel D), B-cells are clearly separated from all other events based on all informative parameters (e.g. CD19, CD3, SSC).

FIG. 4 .—Illustrating example of how to use immunophenotypic characterization markers CD27, CD5, CD22, CD200 and CD79b (panels A to C) for the distinction between CLL cells and normal mature B-cells in a peripheral blood sample from a CLL patient. Only peripheral blood B-cells are displayed, after gated/selected as described in FIG. 3 . In each plot, black dots correspond to CLL cells in the sample, while grey dots are normal peripheral blood B-cells. Compared to normal B-cells, CLL cells show underexpression of CD22 (Panel B) and CD79b (Panel C) together with overexpression of CD200 (Panel B) and CD5 (Panels A and C). In the APS1 (principal component 1 vs. principal component 2) representation based on all immunophenotypic markers and scatter characteristics evaluated (Panel D), CLL cells are clearly discriminated from normal B-cells, while this degree of discrimination could not be achieved based on individual markers.

FIG. 5 .—Illustrating example of how to use the CD38 and CD138 identification markers in combination with SSC (Panel A to C) for the distinction between plasma cells and other nucleated cells in a bone marrow sample of an MM patient after therapy. In each plot, grey dots correspond to non-plasma cells in the sample, while black dots are total bone marrow plasma cells. In the multivariate analysis representation (APS1) of principal component 1 vs. principal component 2 (Panel D), plasma cells (black dots) are clearly separated from all other events (grey dots) based on all informative parameters (e.g. CD138, CD38, SSC).

FIG. 6 .—Illustrating example of how to use immunophenotypic characterization markers CD81, CD19, CD45, CD56, CD27, CD117 and CD38, in combination with SSC (Panels A to D) for the distinction between myeloma/malignant plasma cells and normal residual plasma cells in a bone marrow sample of an MM patient after therapy. Only bone marrow plasma cells are displayed, after gated/selected as described in FIG. 5 . In each plot, black dots correspond to myeloma/clonal plasma cells in the sample, while grey dots are normal residual bone marrow plasma cells. Compared to normal plasma cells, myeloma/clonal plasma cells show underexpression of CD81, CD19, CD45, CD27 and CD38 together with higher SSC and overexpression of CD56 and CD117. In the APS1 (principal component 1 vs. principal component 2) representation (Panel E) based on all immunophenotypic markers and scatter characteristics evaluated normal residual plasma cells (grey dots) are clearly discriminated from myeloma/malignant plasma cells (black dots), while this degree of discrimination could not be achieved based on individual markers.

EXPERIMENTAL SECTION

The power of the EuroFlow approach disclosed herein is based on the combination of sets of markers and the usage of multivariate analyses for both the identification of normal cells (e.g. normal precursor B-cells, normal B-lymphocytes and normal plasma cells) and the distinction between normal/reactive cells vs. clonal/malignant cells. For this purpose, a powerful multivariate analysis of the contribution of individual markers for inclusion and exclusion of each marker in the panel according to its contribution over all other markers in the combination. Such a strategy was used to evaluate the selected combinations of most discriminating markers in multiple sequential rounds of experimental testing. Because of this the final proposed antibody combinations became extremely strong when used in combination with the principal component analysis, specifically with the automated population separation (APS) tool of the Infinicyt software, so that the added (independent) value of each marker is used in a single step of analysis.

Herewith we provided the summary of the results of the extensive experimental studies for MRD detection in blood and bone marrow of patients with BCP-ALL (Example 1), CLL (Example 2) and Multiple Myeloma (Example 3).

In the Examples below, lists of markers are provided together with the most frequent phenotypic aberration of these markers in case of BCP-ALL, CLL, and MM/PCD. However, it should be noted that the real discrimination power between normal and malignant cells is based on combinations of markers in the corresponding n-dimensional space, as is clearly visible in the principle component analyses in the figures of Examples 1 to 3. In fact, minor differences of several markers add up to a larger difference in principle component analysis. Therefore the current invention is not about single marker studies for MRD detection, but about carefully selected sets of markers, that allow excellent discrimination between normal cells and their malignant counterparts, such as normal BCP cells vs. BCP-ALL blasts, normal B-lymphocytes vs. B-CLL cells, and normal plasma cells vs. MM/PCD plasma cells.

Example 1. Antibody Panels and Diagnostic Method for MRD Detection in BCP-ALL Patients

Markers for Identification of Total B Cells and B-Cell Precursors in the Bone Marrow

List of relevant identification markers: CD19, CD45

How to use them: Pre-gating using the CD19 marker is essential for identifying a pure B-cell population. To focus on normal B-cell precursors (BCP), CD45-negativity or weak positivity can be used to discriminate BCP from CD45-positive mature B-cells. In case of CD19-directed therapies, CD19 might be replaced by CD22. These markers may be used in combination also with sideward light scatter (SSC) or forward light scatter (FSC) or both FSC and SSC to identify B-cells in peripheral blood or bone marrow or other types of samples (e.g. bone marrow, tissue biopsy, spinal fluid). Of note, other markers, like CD10, CD20, CD38 and CD34, which are used for discriminating BCP-ALL cells from normal BCP cells (see below), may also contribute to the gating of the total BCP cell population (e.g. CD34+, CD10+, CD20-to dim, CD38+).

Markers for Distinguishing Normal Vs. Malignant B-Cell Precursor Cells

List of markers and most frequent phenotypic aberration:

-   -   CD38: underexpressed in BCP-ALL/malignant vs. normal B-cell         precursor cells     -   CD10: over- or underexpressed in BCP-ALL/malignant B-cell         precursor cells     -   CD45: underexpressed (usually negative) in BCP-ALL/malignant vs.         normal B-cell precursor cells     -   CD20: under- or overexpressed in BCP-ALL/malignant vs. normal         B-cell precursor cells     -   CD81: over- or underexpressed in BCP-ALL/malignant vs. normal         B-cell precursor cells     -   CD66c: overexpressed in BCP-ALL/malignant vs. normal B-cell         precursor cells (particularly BCR-ABL positive ALL; generally         negative in TEL-AML1-positive or MLL-AF4-positive ALL)     -   CD123: overexpressed in BCP-ALL/malignant vs. normal B-cell         precursor cells (particularly in hyperdiploid ALL)     -   CD304: overexpressed in BCP-ALL/malignant vs. normal B-cell         precursor cells     -   CD73: overexpressed in BCP-ALL/malignant vs. normal B-cell         precursor cells     -   CD34: under- or overexpressed in BCP-ALL/malignant vs. normal         B-cell precursor cells     -   SSC: increased or decreased intensity in BCP-ALL/malignant vs.         normal B-cell precursor cells.     -   FSC: increased or decreased intensity in BCP-ALL/malignant vs.         normal B-cell precursor cells.

Example 2. Antibody Panels and Diagnostic Method for MRD Detection in CLL Patients

Markers for Identification of Total B-Cells in Peripheral Blood and Bone Marrow:

List of identification markers: CD19, CD3 (exclusion marker)

How to use them: Pre-gating using this marker combination is essential for identifying a pure B-cell population, and removing T-cell/B-cell doublets. These markers may be used in combination also with sideward light scatter (SSC) or forward light scatter (FSC) or both FSC and SSC to identify B-cells in peripheral blood or bone marrow or other types of samples (e.g. tissue biopsy, spinal fluid). For a more refined gating with better enrichment of CLL cells, both CD5 and CD27 may be used.

Markers for Distinguishing Normal B-Cells from CLL Cells:

List of markers and most frequent phenotypic aberration:

-   -   CD27: positive on CLL cells and a small fraction of normal         B-cells     -   CD5: positive on CLL cells and a small fraction of normal         B-cells     -   CD79b: underexpressed on CLL cells as compared to normal         transitional and mature B-lymphocytes     -   CD22: underexpressed on CLL cells as compared to normal         transitional and mature B-lymphocytes     -   CD20: underexpressed on CLL cells as compared to normal         transitional and mature B-lymphocytes     -   CD200: overexpressed on CLL cells as compared to normal         transitional and mature B-lymphocytes     -   ROR1: overexpressed on CLL cells as compared to normal         transitional and mature B-lymphocytes     -   CD43: overexpressed on CLL cells as compared to normal         transitional and mature B-lymphocytes     -   CD81: underexpressed on CLL cells as compared to B-cell         precursors and both transitional and mature B-lymphocytes     -   CD38: underexpressed on CLL cells as compared to B-cell         precursors

Example 3. Antibody Panels and Diagnostic Method for MRD Detection in MM/PCD Patients

Markers for Identification of Total Plasma Cells in the Bone Marrow:

List of identification markers: CD38, CD138 and CD229

How to use them: Any combination of the three markers in any fluorochrome position works; also it is possible to use any combinations of two of the three markers or in a subset of cases (not all) even one of the three markers alone. Preferable combinations are order as follows: 1) CD138/CD38/CD229; 2) CD138/CD38, 3) CD138/CD229; 4) CD38/CD229; 5) CD138; 6) CD38); 7) CD229. Note that any of these markers individually and in combination may be used in combination also with sideward light scatter (SSC) or forward light scatter (FSC) or both FSC and SSC to identify plasma cells in the bone marrow or other types of samples (e.g. peripheral blood, tissue biopsy, spinal fluid).

Markers for Distinguishing Normal Vs Clonal/Malignant Plasma Cells:

List of markers and most frequent phenotypic aberration:

-   -   CD38: underexpressed in malignant plasma cells compared to         normal plasma cells     -   CD27: underexpressed in malignant plasma cells compared to         normal plasma cells     -   CD45: underexpressed in malignant plasma cells compared to         normal plasma cells     -   CD19: underexpressed (usually negative) in malignant plasma         cells compared to normal plasma cells     -   CD81: underexpressed in malignant plasma cells compared to         normal plasma cells     -   CD56: overexpressed in malignant plasma cells compared to normal         plasma cells     -   CD28: overexpressed in malignant plasma cells compared to normal         plasma cells     -   CD117: overexpressed in malignant plasma cells compared to         normal plasma cells     -   CyIgk and CyIglambda: expression restricted to either one or the         other Ig light chains in malignant plasma cells while showing a         balanced distribution     -   (CyIgk/CyIglambda ratio in normal plasma cells usually ranging         between ratios 3 and 0.5).     -   SSC: increased or decreased intensity in malignant plasma cells         compared to normal plasma cells.     -   FSC: increased or decreased intensity in malignant plasma cells         compared to normal plasma cells. 

The invention claimed is:
 1. A reagent composition for flow cytometric detection of B-cell precursor ALL (BCP-ALL) in a human subject, comprising a panel of at least eight distinct fluorochrome-conjugated antibodies, the panel comprising at least antibodies against the core markers CD10, CD19, CD20, CD34 and CD45, and wherein the panel further comprises one or more antibodies selected from the group consisting of antibodies against CD38, CD81, CyIgμ, and deoxynucleotidyl transferase (NuTdT), and wherein the panel further comprises one or more sets of antibodies selected from the group consisting of: (a) set of antibodies against CD66c and CD123; (b) set of antibodies against CD304 and CD73; and (c) set of antibodies against SmIgκ and SmIgλ, wherein the antibodies within each set are conjugated to the same fluorochrome and wherein between different sets the fluorochromes are distinguishable.
 2. The reagent composition according to claim 1, wherein the panel comprises two or more sets of antibodies selected from the group consisting of: (a) set of antibodies against CD66c and CD123; (b) set of antibodies against CD304 and CD73; and (c) set of antibodies against SmIgκ and SmIgλ.
 3. The reagent composition according to claim 1, comprising distinct fluorochrome-conjugated antibodies directed against one of the following combinations of markers: (i) CD20, CD45, CD81, CD66c, CD123, CD34, CD19, CD10 and CD38, wherein the antibodies against CD66c and CD123 are conjugated to the same fluorochrome; (ii) CD20, CD45, CD81, CD304, CD73, CD34, CD19, CD10 and CD38, wherein the antibodies against CD304 and CD73 are conjugated to the same fluorochrome; or (iii) CD20, CD45, NuTdT, SmIgκ, SmIgλ, CyIgμ, CD19, CD34 and CD10, wherein the antibodies against SmIgκ and SmIgλ are conjugated to the same fluorochrome.
 4. The reagent composition according to claim 1, comprising distinct fluorochrome-conjugated antibodies directed against the markers CD20, CD45, CD81, NuTdT, CD34, CD19, CD10 and CD38, and one or more sets of antibodies selected from the group consisting of: (a) set of antibodies against CD66c and CD123; (b) set of antibodies against CD304 and CD73; and (c) set of antibodies against SmIgκ and SmIgλ, wherein the antibodies within each set are conjugated to the same fluorochrome.
 5. The reagent composition according to claim 4, comprising distinct fluorochrome-conjugated antibodies directed against the markers CD20, CD45, CD81, NuTdT, CD66c, CD123, CD304, CD73, SmIgκ, SmIgλ, CyIgμ, CD34, CD19, CD10 and CD38, wherein the antibodies against each of the sets CD66c/CD123, CD304/CD73 and SmIgκ/SmIgλ are conjugated to the same fluorochrome.
 6. A diagnostic kit for flow cytometric detection of B cell precursor ALL (BCP-ALL), comprising at least one reagent composition according to claim 1, optionally together with instructions for use, buffer, and/or control samples.
 7. The diagnostic kit according to claim 6, wherein the antibody against CD20 is conjugated to PacB; the antibody against CD45 is conjugated to PacO; the antibody against CD81 is conjugated to FITC; the antibody against NuTdT is conjugated to FITC; the antibody against CD66c and the antibody against CD123 are conjugated to PE; the antibody against CD304 and the antibody against CD73 are conjugated to PE; the antibody against SmIgκ and the antibody against SmIgλ are conjugated to PE; the antibody against CD34 is conjugated to PerCPCy5.5; the antibody against CyIgμ is conjugated to PerCPCy5.5; the antibody against CD19 is conjugated to PECy7; the antibody against CD10 is conjugated to APC or APCC750; and the antibody against CD38 is conjugated to APCC750.
 8. A multi-color flow cytometric method for detecting B cell precursor ALL (BCP-ALL) in a biological sample comprising cells, preferably lymphocytes, comprising the steps of: (i) staining the sample with a reagent composition according to claim 1, (ii) subjecting the sample to flow cytometry; (iii) gating on cells for expression of the selected markers detected by the antibodies present in the reagent composition; and (iv) distinguishing between normal and malignant cells, based on the expression profile of the multiple markers.
 9. The method according to claim 8, wherein step (iv) involves multivariate analysis, optionally principal component analysis (PCA).
 10. The multi-color flow cytometric method according to claim 8, wherein the antibody against CD20 is conjugated to PacB; the antibody against CD45 is conjugated to PacO; the antibody against CD81 is conjugated to FITC; the antibody against NuTdT is conjugated to FITC; the antibody against CD66c and the antibody against CD123 are conjugated to PE; the antibody against CD304 and the antibody against CD73 are conjugated to PE; the antibody against SmIgκ and the antibody against SmIgλ are conjugated to PE; the antibody against CD34 is conjugated to PerCPCy5.5; the antibody against CyIgμ is conjugated to PerCPCy5.5; the antibody against CD19 is conjugated to PECy7; the antibody against CD10 is conjugated to APC or APCC750; and the antibody against CD38 is conjugated to APCC750.
 11. The reagent composition according to claim 1, wherein the antibody against CD20 is conjugated to PacB; the antibody against CD45 is conjugated to PacO; the antibody against CD81 is conjugated to FITC; the antibody against NuTdT is conjugated to FITC; the antibody against CD66c and the antibody against CD123 are conjugated to PE; the antibody against CD304 and the antibody against CD73 are conjugated to PE; the antibody against SmIgκ and the antibody against SmIgλ are conjugated to PE; the antibody against CD34 is conjugated to PerCPCy5.5; the antibody against CyIgμ is conjugated to PerCPCy5.5; the antibody against CD19 is conjugated to PECy7; the antibody against CD10 is conjugated to APC or APCC750; and the antibody against CD38 is conjugated to APCC750. 